br EGFR a transmembrane tyrosine
EGFR, a transmembrane tyrosine kinase, transmits important
growth factor signals of the extracellular milieu into cells. More than 60% of NSCLCs express EGFR, which is an important therapeutic target for the treatment of NSCLCs [47,48]. CAP and iron oxide-based MNPs either individually or in combination significantly reduced mRNA and protein expression levels of EGFR (Fig. 8). EGFR and its downstream signaling pathway are involved in the proliferation of cancer, the ac-tivation of AKT and ERK1/2 are also participate in the proliferation and diﬀerentiation, treatment of CAP to cancer Chloramphenicol could inactive AKT and ERK1/2 [49,50]. Expression of EGFR in cancer cell is regulated by TGF-α, E-cadherin and some miRNAs, although our results showed that E-cadherin were downregulated in CAP and MNPs cotreated lung cancer cells, whether CAP and MNPs could modulated the expression of EGFR via TGF-α and CDH1 and the detail underlying mechanisms need fur-ther exploration. EGFR-RAS/ERK and EGFR-PI3K/AKT signaling path-ways are essential for the regulation of cellular proliferation and are implicated in tumor initiation and development [51,52]. Previously research showed that pERK 1/2 and pAKT expression levels were de-clined by plasma-activated medium (PAM) in glioblastoma cells [53,54]. We characterized the eﬀect of CAP and iron oxide-based MNPs on EGFR-RAS/ERK and EGFR-PI3K/AKT pathways. The expression le-vels of pERK and pAKT were significantly decreased after co-treatment of CAP and iron oxide-based MNPs. The co-treatment of CAP and iron oxide-based MNPs inhibited cell growth through the downregulation of pERK and pAKT, which are important phosphokinases in tumorigenesis and tumor progression. EGFR-RAS/ERK and EGFR-PI3K/AKT signaling pathways are related to EMT [55,56], the combination of CAP and iron
Fig. 9. Iron oxide-based MNPs and CAP attenuates tumor growth in xenograft nude mice models: (a) Growth curves of xenograft NSCLC tumors in nude mice treated by CAP and iron oxide-based MNPs, (b) Tumor bearing mice co-treated with MNPs and CAP. (c) Weight changes of xenograft tumor. (d) HE staining of tumor tissue sections (up panel), Tunel analysis of apoptosis in treated and untreated tumors tissues (middle panel), Prussian blue staining show the distribution of Fe ions (down panel). (e) Immunohistochemistry analysis showed the expression changes of E-cadherin, vimentin, EGFR and Ki-67 in treated and untreated tumors.
oxide-based MNPs promoted A549 cancer cell death and reversed EMT (Fig. 7). Further experiments revealed the upregulation of E-cadherin and the downregulation of vimentin, emphasizing the reversal of EMT. Moreover, the anti-tumor capabilities of CAP and iron oxide-based MNPs on tumor in vivo were confirmed (Fig. 9). The combination of CAP and iron oxide-based MNPs treating the implanted tumors in mice remarkably reduced the tumor size. The application of CAP to issues is a multi-phase process, resulting into a diﬀusive interface with a liquid-like layer or environment. There is a boundary plasma in the surface of biological tissue. Species in CAP lead to the creation of various ROS across the plasma-liquid interface and their propagation towards and diﬀusion through an arbitrary tissue layer . On the other hand, Yoon et al. indicated that CAP could penetrate around 2 mm from skin to tumor . In our mouse model, the thickness is less than 2 mm from skin to tumor. Thus, CAP can penetrate skin to tumor, inducing new ROS generation in tumor. We consider that CAP-derived ROS or CAP-induced ROS generation in tumor contribute to CAP and MNPs syner-getic eﬀects in the xenograft models. However, penetrability of CAP is still a big challenge for plasma application, more work should be done in future to indicate mechanism of plasma penetrability.
Immunohistochemical staining revealed the abolished vimentin levels and increased E-cadherin levels in the tissues subjected to dual treat-ment. Overall, H2O2 was generated in CAP-treated A549 cancer cells [59,60], while Fe2+/Fe3+ from iron oxide-based MNPs can transform H2O2 into •OH via Fenton’s reaction . Iron oxide-based MNPs can enhance the tumor-killing eﬀect of CAP through this mechanism (Fig. 10). These results suggested that dual treatment with CAPs and iron oxide-based MNPs potentially inhibited tumor growth.
In summary, the eﬀect of co-treatment of CAP and iron oxide-based MNPs both in vitro and in vivo has been demonstrated. The co-treat-ment could eﬀectively induce A549 cancer cells apoptosis in vitro via the inhibition of EGFR-RAS/ERK and EGFR-PI3K/AKT signaling path-ways and the modulation of EMT. The co-treatment also suppressed xenograft tumor growth. Thus, the combination of CAP and iron oxide-based MNPs may lead to a shift in the paradigm of cancer therapy.